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• W2034004569 abstract "We demonstrated that rat kinin B1 receptors displayed a ligand-independent constitutive activity, assessed through inositol phosphate production in transiently or stably transfected human embryonic kidney 293A cells. Substitution of Ala for Asn130 in the third transmembrane domain resulted in additional constitutive activation of the B1 receptor. The constitutively active mutant N130A receptor could be further activated by the B1 receptor agonist des-Arg9-bradykinin. To gain insights into the physiological function of the B1 receptor, we have generated transgenic mice overexpressing wild-type and constitutively active mutant receptors under the control of human cytomegalovirus immediately early gene enhancer/promoter. The rat B1receptor transgene expression was detected in the aorta, brain, heart, lung, liver, kidney, uterus, and prostate of transgenic mice by reverse transcription-polymerase chain reaction/Southern blot analysis. Transgenic mice were fertile and normotensive. Overexpression of B1 receptors exacerbated paw edema induced by carrageenan and rendered transgenic mice more susceptible to lipopolysaccharide-induced endotoxic shock. Interestingly, the hemodynamic response to kinins was altered in transgenic mice, with des-Arg9-bradykinin inducing blood pressure increase when intravenously administered. Our study supports an important role for B1 receptors in modulating inflammatory responses and for the first time demonstrates that B1 receptors mediate a hypertensive response to des-Arg9-bradykinin. We demonstrated that rat kinin B1 receptors displayed a ligand-independent constitutive activity, assessed through inositol phosphate production in transiently or stably transfected human embryonic kidney 293A cells. Substitution of Ala for Asn130 in the third transmembrane domain resulted in additional constitutive activation of the B1 receptor. The constitutively active mutant N130A receptor could be further activated by the B1 receptor agonist des-Arg9-bradykinin. To gain insights into the physiological function of the B1 receptor, we have generated transgenic mice overexpressing wild-type and constitutively active mutant receptors under the control of human cytomegalovirus immediately early gene enhancer/promoter. The rat B1receptor transgene expression was detected in the aorta, brain, heart, lung, liver, kidney, uterus, and prostate of transgenic mice by reverse transcription-polymerase chain reaction/Southern blot analysis. Transgenic mice were fertile and normotensive. Overexpression of B1 receptors exacerbated paw edema induced by carrageenan and rendered transgenic mice more susceptible to lipopolysaccharide-induced endotoxic shock. Interestingly, the hemodynamic response to kinins was altered in transgenic mice, with des-Arg9-bradykinin inducing blood pressure increase when intravenously administered. Our study supports an important role for B1 receptors in modulating inflammatory responses and for the first time demonstrates that B1 receptors mediate a hypertensive response to des-Arg9-bradykinin. transmembrane domain G protein-coupled receptor bradykinin des-Arg9-BK lipopolysaccharide inositol phosphates mean arterial blood pressure Kinin peptides are released from kininogen precursors by the action of kallikreins in response to tissue injury (1Bhoola K.D. Figueroa C.D. Worthy K. Pharmacol. Rev. 1992; 44: 1-80PubMed Google Scholar). Kinins induce smooth muscle contraction, vasodilation, increased vascular permeability, and pain (1Bhoola K.D. Figueroa C.D. Worthy K. Pharmacol. Rev. 1992; 44: 1-80PubMed Google Scholar). Kinins exert their effects through selective activation of two seven-transmembrane domain (TMD)1 G protein-coupled receptors (GPCRs), B1 and B2 (2Regoli D. Barabe J. Pharmacol. Rev. 1980; 32: 1-46Crossref PubMed Scopus (16) Google Scholar, 3Hess J.F. Borkowski J.A. Young G.S. Strader C.D. Ransom R.W. Biochem. Biophys. Res. Commun. 1992; 184: 260-268Crossref PubMed Scopus (448) Google Scholar, 4Menke J.G. Borkowski J.A. Bierilo K.K. MacNeil T. Derrick A.W. Schneck K.A. Ransom R.W. Strader C.D. Linemeyer D.L. Hess J.F. J. Biol. Chem. 1994; 269: 21583-21586Abstract Full Text PDF PubMed Google Scholar). The B2 receptor is constitutively expressed, mediating the actions of intact kinins, bradykinin (BK) in rodents and Lys-BK or kallidin in humans (2Regoli D. Barabe J. Pharmacol. Rev. 1980; 32: 1-46Crossref PubMed Scopus (16) Google Scholar). In contrast, the B1 receptor is expressed at very low levels in normal tissues in most animal species but is induced under the influence of inflammation or exposure of tissues to noxious stimuli, mediating the effects of the carboxypeptidase metabolites of intact kinins, des-Arg9-BK (DABK), and des-Arg10-kallidin (2Regoli D. Barabe J. Pharmacol. Rev. 1980; 32: 1-46Crossref PubMed Scopus (16) Google Scholar). The cellular responses of kinin receptors to agonists are transduced primarily via coupling to either Gq protein, which in turn activates phospholipase C to stimulate inositol phosphate production, or the Giprotein, acting through phospholipase A2 to stimulate arachidonic acid pathway (5Schneck K.A. Hess J.F. Stonesifer G.Y. Ransom R.W. Eur. J. Pharmacol. 1994; 266: 277-282Crossref PubMed Scopus (57) Google Scholar, 6Tropea M.M. Gummelt D. Herzig M.S. Leeb-Lundberg L.M. J. Pharmacol. Exp. Ther. 1993; 264: 930-937PubMed Google Scholar). Over the past years, transgenic and gene-targeting technologies associated with molecular biology tools have provided important knowledge concerning the role of kinin receptors in vivo. Transgenic mice expressing the human B2 receptor under the control of the Rous sarcoma virus 3′-long terminal repeat promoter were hypotensive compared with control littermates (7Wang D.Z. Chao L. Chao J. Hypertension. 1997; 29: 488-493Crossref PubMed Google Scholar). Administration of the B2 receptor antagonist Hoe-140 blunted the blood pressure-lowering effect of the transgene, whereas intra-arterial bolus injection of BK produced more pronounced blood pressure reduction (7Wang D.Z. Chao L. Chao J. Hypertension. 1997; 29: 488-493Crossref PubMed Google Scholar). In contrast, deletion of the B2 receptor in mice produced an unaltered blood pressure phenotype (8Borkowski J.A. Ransom R.W. Seabrook G.R. Trumbauer M. Chen H. Hill R.G. Strader C.D. Hess J.F. J. Biol. Chem. 1995; 270: 13706-13710Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar) but led to salt-sensitive hypertension and altered nociception (9Rupniak N.M. Boyce S. Webb J.K. Williams A.R. Carlson E.J. Hill R.G. Borkowski J.A. Hess J.F. Pain. 1997; 71: 89-97Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 10Madeddu P. Varoni M.V. Palomba D. Emanueli C. Demontis M.P. Glorioso N. Dessi-Fulgheri P. Sarzani R. Anania V. Circulation. 1997; 96: 3570-3578Crossref PubMed Scopus (149) Google Scholar). Using specific antagonists, the B1 receptor has been implicated in toxic shock, inflammation, and nociception (11Marceau F. Hess J.F. Bachvarov D.R. Pharmacol. Rev. 1998; 50: 357-386PubMed Google Scholar). Studies of mice lacking the B1 receptor provided support to these observations. B1 receptor knockout animals were healthy, fertile, and normotensive and exhibited hypoalgesia and reduced inflammatory response (12Pesquero J.B. Araujo R.C. Heppenstall P.A. Stucky C.L. Silva J.A., Jr. Walther T. Oliveira S.M. Pesquero J.L. Paiva A.C. Calixto J.B. Lewin G.R. Bader M. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8140-8145Crossref PubMed Scopus (328) Google Scholar). Although much has been learned about the physiological role of the B1 receptor, most studies are about the lipopolysaccharide (LPS)-induced B1 receptors, because the B1receptor is expressed at very low levels, if at all, in normal tissue. In such experimental set-ups, the animals are under systemic inflammation conditions, which preclude the direct study of the function of the B1 receptor. Therefore, the precise physiological and pathophysiological roles of the B1receptor remain elusive. To extend our understanding of the physiological function of the B1 receptor, we have created a constitutively active mutant of the rat B1 receptor and generated transgenic mice that overexpress the wild-type B1receptor and the mutant receptor under the control of human cytomegalovirus immediately early gene enhancer/promoter. The transgenic mice were characterized, and the findings are reported here. Human embryonic kidney 293A cells were obtained from Quantum Biotechnologies. LPS (Salmonella enteritidis, LD50: 7.20 mg/kg) was from Difco Laboratories. myo-[3H]Inositol was from PerkinElmer Life Sciences. LipofectAMINE, culture medium, restriction enzymes, and fetal calf serum were bought from Invitrogen. Puromycin was from Clontech Lab. Hoe-140 was obtained from Hoechst. Sar-Tyr-εAhx-Lys-des-Arg9-bradykinin and Sar-Tyr-εAhx-Lys-[d-βNal7,Ile8]-des-Arg9-bradykinin were gifts from Dr. D. Regoli (13Gobeil F. Neugebauer W. Nguyen-Le X.K. Nea Allogho S. Pheng L.H. Blouin D. Whalley E.T. Regoli D. Can. J. Physiol. Pharmacol. 1997; 75: 591-595Crossref PubMed Google Scholar). All other chemicals were purchased from Sigma unless stated otherwise. The mutations were created by using the QuikChange site-directed mutagenesis kit (Stratagene), and the previously cloned wild-type rat B1 cDNA in pcDNA3 (Invitrogen) was used as a template (14Ni A. Chai K.X. Chao L. Chao J. Biochim. Biophys. Acta. 1998; 1442: 177-185Crossref PubMed Scopus (62) Google Scholar). The following oligonucleotides were used as forward primers: 5′-GGC CTC TTG GGGGCC CTT TTA GTC TTG TC-3′ for the preparation of clone N54A (nucleotide changes underlined), 5′-GTC ATC AAG GCC GCC CTG TTT GTC AG-3′ for clone N130A, 5′-GCT ATC AGT CAGCAA CGC TAC AGG CTC-3′ for clone D144Q, and 5′-GCT ATC AGT CAG ACC CGC TAC AGG CTC-3′ for clone D144T. The reverse primers were complementary to the forward primers described. All of the mutations were confirmed by DNA sequencing. TheNruI/NotI fragments in the resultant plasmids were released and inserted at NruI/NotI sites in pIRESpuro (Clontech Lab), leading to vectors for stable transfection. For transient transfection, 293A cells were seeded into 12-well trays and left to adhere overnight. pcDNA3-derived expression vectors were transfected with LipofectAMINE as previously described (14Ni A. Chai K.X. Chao L. Chao J. Biochim. Biophys. Acta. 1998; 1442: 177-185Crossref PubMed Scopus (62) Google Scholar). Transfection mixtures were left on cells for 5 h, and then the cells were treated with a change of standard growth medium for 48 h before functional studies. For stable transfection, 293A cells were transfected with pIRESpuro-derived expression vectors. After 16 h, the medium was changed with complete medium containing puromycin (2 μg/ml) to start the selection of stably transfected cells. The medium was changed every 3 days, and after about 12 days, the colonies surviving selection were lifted into 12-well plates, expanded with a maintenance concentration of 2 μg/ml puromycin, and screened for ligand specific binding. All of the stock cultures were kept under constant selection pressure of 2 μg/ml puromycin, whereas cells seeded in dishes/wells were maintained without puromycin and used within 2–3 days. Monolayers of transfected 293A cells grown in 12-well trays were labeled for 24 h with 2 μCi of [3H]inositol in 0.5 ml of inositol-free Dulbecco's modified Eagles' medium supplemented with 0.05% bovine serum albumin and penicillin/streptomycin. After equilibrated in prewarmed Dulbecco's modified Eagles' medium containing 140 μg/ml bacitracin, 100 μm Captopril, and 25 mm LiCl for 15–30 min, the cells were stimulated with various ligands at the indicated concentrations for 20 min at 37 °C. The released total inositol phosphates (IP) were isolated using Bio-Rad AG1-X8 anion exchange columns (1-ml volume) and quantified as described (15Berridge M.J. Downes C.P. Hanley M.R. Biochem. J. 1982; 206: 587-595Crossref PubMed Scopus (1798) Google Scholar,16Faussner A. Proud D. Towns M. Bathon J.M. J. Biol. Chem. 1998; 273: 2617-2623Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar). Transfected 293A cell monolayers in 12-well plates were washed twice with Dulbecco's modified Eagles' medium and incubated at 4 °C with radioligand [125I]Sar-Tyr-εAhx-Lys-des-Arg9-bradykinin (14Ni A. Chai K.X. Chao L. Chao J. Biochim. Biophys. Acta. 1998; 1442: 177-185Crossref PubMed Scopus (62) Google Scholar) in the presence or absence of 5 μm of the unlabeled ligand in 0.3 ml of Dulbecco's phosphate-buffered saline supplemented with 140 μg/ml bacitracin, 1 mg/ml bovine serum albumin, 1 mm 1,10-phenanthroline, and 100 μm Captopril. The incubation lasted at least 3 h under gentle agitation. The cells were then rinsed twice with ice-cold phosphate-buffered saline with 0.3% bovine serum albumin followed by solubilization in 0.5 ml of 0.1 m NaOH. The radioactivity of the sample was quantified with a 1261 Mutigamma counter (Pharmacia Corp.). The cell number was determined in parallel wells. Stably transfected 293A cells grown in 6-well plates were preincubated for 15 min with Dulbecco's modified Eagles' medium containing 1 mm 3-isobutyl-1-methylxanthine and 100 μm Captopril and then stimulated with 1 μm DABK for 15 min. The stimulation was terminated by exchanging the incubation medium for 0.5 ml of ice-cold 0.1m HCl. The cGMP and cAMP productions were determined by radioimmunoassys as described (17Brooker G. Harper J.F. Terasaki W.L. Moylan R.D. Adv. Cyclic Nucleotide Res. 1979; 10: 1-33PubMed Google Scholar, 18Wang D. Yoshida H. Song Q. Chao L. Chao J. Am. J. Physiol. 2000; 278: F484-F491Google Scholar). The bovine growth hormone poly(A) sequence in pcDNA3 was released with ApaI and PvuII and inserted at ApaI/EcoRV sites in pBluescript KS II (Stratagene). The ApaI/SmaI bovine growth hormone poly(A) fragment was then released and inserted atApaI/SmaI sites in the pcDNA3-derived wild-type and N130A receptor expression vectors described above. The human 4F2 enhancer was amplified by PCR from genomic DNA with the primers 5′-TAC TCG AGT GCA GCG CGC CCC CG-3′ and 5′-CTG GGC CCT TCA CCT TCA GAG AGC-3′ (19Karpinski B.A. Yang L.H. Cacheris P. Morle G.D. Leiden J.M. Mol. Cell. Biol. 1989; 9: 2588-2597Crossref PubMed Scopus (43) Google Scholar). After being cut with XbaI andApaI, the 4F2 enhancer fragment was inserted atXbaI/ApaI sites, resulting in final transgene vectors. The vectors were cut with NruI and SmaI, and the linear transgenes were separated from the unneeded fragments with agarose gel and prepared for injection by Qiaquick gel extraction columns (Qiagen). Transgenic mice were created by the Transgenic Facility at the Medical University of South Carolina and the Transgenic Facility of University of Ohio at Cincinnati. Linear transgene was injected into the pronuclei of one-cell mouse embryos, which were then surgically implanted into pseudopregnant female mice. Transgenic founder mice were identified by Southern blot analysis of genomic DNA isolated from tail biopsies. Positive founders identified from each line were bred with normal mice, and then F1 littermates were crossed between themselves. Tail DNA was digested with restriction enzyme KpnI, run on 0.7% agarose gels containing ethidium bromide, and transferred to Immobilon-N membrane by capillary action with 10× SSC overnight, and the blots were hybridized to a rat kinin B1 receptor cDNA probe as described previously (14Ni A. Chai K.X. Chao L. Chao J. Biochim. Biophys. Acta. 1998; 1442: 177-185Crossref PubMed Scopus (62) Google Scholar,20Chai K.X., Ni, A. Wang D. Ward D.C. Chao J. Chao L. Genomics. 1996; 31: 51-57Crossref PubMed Scopus (46) Google Scholar). Total RNA was extracted from mouse tissues using the RNeasyTM columns (Qiagen). Reverse transcription-PCR/Southern blot analysis was performed using the transgene-specific primers and internal probe as previously described (21Ni A. Chao L. Chao J. J. Biol. Chem. 1998; 273: 2784-2791Abstract Full Text Full Text PDF PubMed Scopus (125) Google Scholar). The upstream primer is 5′-ATG GCG TCC GAG GTC TT-3′; the downstream primer is 5′-GAC AAA CAC CAG ATC GG-3′; and the internal probe is 5′-TGG CAG CAA CGA CAG AG-3′. The mice were sacrificed by cervical dislocation. The kidney was removed, and the wet weight was determined. The tissue was homogenized using a Polytron in ∼20 volumes of ice-cold 20 mm HEPES, pH 7.4. The membranes from the tissue were prepared, and binding assays were performed as described previously (7Wang D.Z. Chao L. Chao J. Hypertension. 1997; 29: 488-493Crossref PubMed Google Scholar, 14Ni A. Chai K.X. Chao L. Chao J. Biochim. Biophys. Acta. 1998; 1442: 177-185Crossref PubMed Scopus (62) Google Scholar). Systolic blood pressure was measured using a computer system RTBP2000 (Kent Scientific) according to the manufacturer's instruction. Briefly, the mice were placed into the prewarmed harness (38 °C). The tail was placed in the occlusion cuff/piezoelectric pulse sensor and distention caused by arterial blood pulses was detected by the sensor and read out onto the computer system. Pressure in the cuff was increased until the pulse was lost. Actual blood pressure was measured as the pressure at which a pulse was detected during cuff depressurization. Ten readings were taken for each animal. The mice were initially anesthetized with 2,2,2-tribromoethanol intert-amyl alcohol (Avertin, 20 mg/ml, 0.4 ml/25 g of body weight) and placed on a heated table to maintain body temperature. The right jugular vein and the left carotid artery were cannulated with PE-l0 catheters (Clay Adams). After the animals were allowed to recover, blood pressure (in the carotid artery) and heart rate were recorded using a computer system MP100 (Biopac systems Inc). The mice were given a bolus injection of BK or DABK from right jugular vein. BK or DABK was serially diluted and administered at doses of 75, 150, and 300 ng in a volume of 50 μl of saline/mouse. Inflammation of one hind paw of mice was induced by intraplantar injection of 20 μl of 1% carrageenan (dissolved in saline), 3 μg of capsaicin in 10 μl (dissolved in 5% ethanol, 5% Tween 80 and 90% saline), or DABK (50 or 300 nmol in saline), whereas the contralateral paw received the same volume of vehicle. Thirty min post injection of capsaicin and DABK or 3 h after carrageenan administration, the mice were sacrificed, both hind paws were cut off at the ankle, and the difference between their weights, representing paw edema, was calculated. LPS was dissolved in sterile 0.9% NaCl. The mice were injected intraperitoneally with a single dose of LPS (24 mg/kg body weight), and the percentage of survivors was determined at 12-h intervals. Both of the control groups were injected with 0.9% NaCl. The group data are expressed as the means ± S.E. The data were compared between experimental groups by one-way analysis of variance. Differences between groups were further evaluated by Fisher's protected least squares differences. Differences were considered significant at a value of p< 0.05. To generate the constitutively active mutants of B1 receptors, site-directed mutagenesis was directed at the Asn54, Asn130, and Asp144 residues in the rat B1 receptor (Fig. 1). The amino acid replacements were N54A, N130A, D144Q, and D144T. The receptor expression vectors were transiently transfected into 293A cells for assessing constitutive activity by determining agonist-independent IP production. At optimal transfection conditions, all of the mutants were expressed at a comparable level but significantly lower than the wild-type receptor (data not shown). As shown in Table I, the wild-type B1 receptor displayed a marked ligand-independent, spontaneous activity (104% above control levels of the mock-transfected cells), and the N54A mutant showed an impaired basal activity compared with the wild-type receptor. Substitution of Ala for Asn130 resulted in significantly constitutive activation of the rat B1 receptor (409% above control levels). In contrast, the agonist-independent IP accumulations in 293A cells expressing D144Q and D144T mutants were similar to that of mock transfected cells, and the constitutive activity of the wild-type receptor was abolished by the mutations. Interestingly, the maximal extent of DABK stimulation of IP production was significantly reduced for all mutants compared with the wild-type receptor.Table IIP accumulation in 293A cells expressing wild-type or mutant receptorsReceptor[3H]IP accumulationR max1-aThe R maxvalues are percentages above basal.Basal IP1-bThe basal IP values are percentages above control.WT1487 ± 112104 ± 27N54A433 ± 3510 ± 4N130A430 ± 18409 ± 23D144Q67 ± 70D144T20 ± 90R max indicates the percentage increase in the 1 μm DABK-induced IP accumulation above basal levels in the absence of DABK. Basal IP indicates the percentage of increase in IP concentrations over those of mock-transfected cells (control) in the absence of DABK. The expression levels of the receptors were comparable except for the wild type (WT) with a higher level of expression. The results are the means ± S.E. of three independent experiments, each performed in duplicate.1-a The R maxvalues are percentages above basal.1-b The basal IP values are percentages above control. Open table in a new tab R max indicates the percentage increase in the 1 μm DABK-induced IP accumulation above basal levels in the absence of DABK. Basal IP indicates the percentage of increase in IP concentrations over those of mock-transfected cells (control) in the absence of DABK. The expression levels of the receptors were comparable except for the wild type (WT) with a higher level of expression. The results are the means ± S.E. of three independent experiments, each performed in duplicate. To better characterize the N130A mutant receptor, stable expression of the N130A mutant in 293A cells was established. For comparison, stable expression of the wild-type rat B1 receptor in 293A cells was also established. Such stably transfected 293A cells were analyzed for receptor density and their affinity for radioligand [125I]Sar-Tyr-εAhx-Lys-des-Arg9-bradykinin and used in functional assays. The N130A receptor was expressed at a level of about 25% of the wild-type receptor in the stably transfected 293A cells (Table II). The maximum number of N130A receptor-binding sites (B max) using [125I]Sar-Tyr-εAhx-Lys-des-Arg9-bradykinin is 1.4 × 105 sites/cell versus 5.3 × 105 sites/cell for the wild-type receptor. In contrast, the mutation of Asn130 into Ala significantly increased the affinity for [125I]Sar-Tyr-εAhx-Lys-des-Arg9-bradykinin. The dissociation constant (K d) for the binding of [125I]Sar-Tyr-εAhx-Lys-des-Arg9-bradykinin by the wild-type and N130A receptors in intact stably transfected 293A cells were 2.54 ± 0.40 and 1.63 ± 0.18 nm, respectively.Table IICharacterization of the wild-type and N130A mutant receptorsReceptorK dB maxcAMPcGMPBasalMaximalBasalMaximalnm(sites/cell × 10 −3 )pmol/mgfmol/mgWT2.54 ± 0.4053016 ± 21858 ± 55421 ± 292208 ± 218N130A1.63 ± 0.1814043 ± 7608 ± 53528 ± 21872 ± 14293A cells12 ± 113 ± 5454 ± 14461 ± 35293A cells stably expressing the wild-type (WT) and the N130A mutant (N130A) rat kinin B1 receptor were established, and the receptor binding sites (B max) and dissociation constant (K d) were determined by using radioligand [125I]Sar-Tyr-ɛAhx-Lys-des-Arg9-bradykinin as described under “Experimental Procedures.” The basal (Basal) and 1 μm DABK-stimulated (Maximal) intracellular cAMP and cGMP levels were also measured. The results are the means ± S.E. of three experiments. Open table in a new tab 293A cells stably expressing the wild-type (WT) and the N130A mutant (N130A) rat kinin B1 receptor were established, and the receptor binding sites (B max) and dissociation constant (K d) were determined by using radioligand [125I]Sar-Tyr-ɛAhx-Lys-des-Arg9-bradykinin as described under “Experimental Procedures.” The basal (Basal) and 1 μm DABK-stimulated (Maximal) intracellular cAMP and cGMP levels were also measured. The results are the means ± S.E. of three experiments. To assess the mode of coupling between the B1 receptor and adenylate cyclase and guanylate cyclase and the effect of the mutation of Asn130 into Ala on the DABK-induced cAMP and cGMP production, the intracellular cAMP and cGMP levels were measured in the stably transfected 293A cells. As shown in Table II, the basal cAMP and cGMP levels in the 293A cells stably expressing the wild-type receptor were similar to those in the nontransfected 293A cells, whereas in the 293A cells stably expressing the N130A receptor, the basal cAMP and cGMP levels were elevated. DABK challenge increased intracellular cAMP and cGMP production in the 293A cells stably expressing the wild-type and N130A receptors: 116-fold increase in cAMP levels and 5.2-fold increase in cGMP levels for the wild-type receptor versus14-fold increase in cAMP levels and 1.7-fold increase in cGMP levels for the N130A mutant. Using the stably transfected 293A cells, the dose-dependent DABK stimulation of IP production for the wild-type and N130A receptor was investigated. As shown in Fig. 2, the N130A mutant could be further activated by over 5-fold by saturation doses of DABK, whereas the wild-type receptor could be amplified to a even higher degree (∼16-fold). The properties of some kinins to modulate IP production by the wild-type and N130A receptors were evaluated. As shown in Fig. 3, kinin B2 receptor agonist BK and antagonist Hoe-140 have no effects. Des-Arg9,[Leu8]-bradykinin and Sar-Tyr-εAhx-Lys-[d-βNal7,Ile8]-des-Arg9-bradykinin are human B1 receptor-specific antagonists. Sar-Tyr-εAhx-Lys-[d-βNal7,Ile8]-des-Arg9-bradykinin is still an antagonist for the wild-type rat B1 receptor, whereas des-Arg9,[Leu8]-braykinin becomes a partial agonist (70–80% of DABK), which provides support for the early observation that des-Arg9,[Leu8]-bradykinin has partial agonist activity in a contraction assay of smooth muscle of rat duodenum and ileum (22Paiva A.C. Paiva T.B. Pereira C.C. Shimuta S.I. Br. J. Pharmacol. 1989; 98: 206-210Crossref PubMed Scopus (20) Google Scholar, 23Meini S. Lecci A. Maggi C.A. Br. J. Pharmacol. 1996; 117: 1619-1624Crossref PubMed Scopus (41) Google Scholar). In contrast, Sar-Tyr-εAhx-Lys-[d-βNal7,Ile8]-des-Arg9-bradykinin becomes a partial agonist, and des-Arg9,[Leu8]-BK becomes a potent agonist for the N130A receptor. Using the wild-type and N130A cDNAs, we have constructed two transgenes for development of transgenic mice. The transgene consists of the cytomegalovirus immediately early gene enhancer/promoter, the wild-type or N130A rat B1 receptor cDNA, the human 4F2 enhancer, and the bovine growth hormone poly(A) sequence (Fig. 4). Three transmitting founder lines, including one wild-type line, WT2510, and two N130A lines, N130A58 and N130A2592, were identified by Southern blot analysis of genomic DNA. Heterozygous wild-type and N130A transgenic mice showed no gross phenotypic abnormalities. All of the transgenic mice were fertile. However, mating F1 generation heterozygotes of both N130A lines produced smaller litters compared with the nontransgenic control and WT2510 line, and a non-Mendelian ratio against N130A mice was observed in the offspring. In the following studies heterozygous transgenic mice were used. The distribution of transgene mRNA expression in F1 and F2 generation heterozygous mice was determined by reverse transcription-PCR/Southern blot analysis. As expected, both male and female transgenic mice showed significant overexpression of the transgene mRNA in the aorta, kidney, liver, heart, brain, and lung and in the prostate of males and the uterus of females. Fig. 5 shows the result from line N130A58. Using specific B1 receptor radioligand [125I]Sar-Tyr-εAhx-Lys-des-Arg9-BK (14Ni A. Chai K.X. Chao L. Chao J. Biochim. Biophys. Acta. 1998; 1442: 177-185Crossref PubMed Scopus (62) Google Scholar), strong B1 receptor binding activity was detected in membranes prepared from the kidneys of all transgenic lines (data not shown). In contrast, neither transgene mRNA expression nor B1 receptor (including endogenous) binding activity could be detected in the corresponding tissues of the nontransgenic control mice. All of the heterozygous transgenic mice were normotensive. The systolic blood pressures of transgenic mice 10 weeks old were 78.7 ± 8.5 mmHg (n = 11) for line WT2510, 75.0 ± 7.6 mmHg (n = 14) for line N130A58, and 76.2 ± 6.3 mmHg (n = 11) for line N130A2592 versus 80.9 ± 3.9 mmHg (n = 11) for age-matched nontransgenic control littermates. Intravenous injection of B1 receptor agonist DABK via the jugular vein produced a transient increase of mean arterial blood pressure (MABP) in anesthetized transgenic mice but not in nontransgenic control littermates (Fig. 6 A). The duration of MABP increase lasted over 5 min. 75 ng of DABK led to an increase of blood pressure by up to 15 mmHg. In contrast, intravenous injection of B2 receptor agonist BK into transgenic mice caused a remarkable primary MABP reduction, followed by a blood pressure bounce-back going beyond basal level, whereas in nontransgenic mice the blood pressure just returned to basal level, after a similar primary MABP reduction (Fig. 6 B). Single or subsequent injections of higher doses of DABK did not result in a further increase of blood pressure in transgenic mice (data not shown). Intraplantar injection of carrageenan resulted in a marked inflammation seen by paw swelling in normal and transgenic mice (Fig. 7). But the paw edema induced in transgenic mice was more severe. The percentage of the weight increase of the carrageenan-injected paw over the contralateral vehicle-injected paw was 38.4 ± 6.7% for" @default.
• W2034004569 created "2016-06-24" @default.
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• W2034004569 date "2003-01-01" @default.
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• W2034004569 title "Overexpression of Kinin B1 Receptors Induces Hypertensive Response to Des-Arg9-bradykinin and Susceptibility to Inflammation" @default.
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